Simplified use of 5&#39; ends of RNAs for cloning and cDNA library construction

ABSTRACT

A method of directional cloning using the 5′ ends of RNAs for use, for example, in cloning and cDNA library construction is provided.

[0001] This application is a continuation of U.S. Ser. No. 09/194,513filed Feb. 25, 1999, which is the U.S. National Phase of PCT/US97/06957filed Apr. 25, 1997, which claims the benefit of U.S. ProvisionalApplication No. 60/016,617 filed May 1, 1996, each of which are hereinincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] Cloning of DNA sequences encoding expressed proteins andconstruction of cDNA libraries from poly A+ mRNAs isolated form cellsand tissues is currently performed in accordance with proceduresoutlined in FIGS. 1a and 1 b. However, the overall process is verylaborious and has several technical limitations. Decreasing activity ofthe reverse transcriptase enzyme during first strand synthesis of thereverse complementary DNA (−) strand from the mRNA can result in yieldof a product that is not full length. In addition, truncations can occurduring the second round of synthesis to regenerate the corresponding“sense” coding (+) DNA sequences from the (−) DNA strand. For abundantmRNAs, a complete full length second strand is not always required asthere is a greater likelihood for overlapping cDNAs that span a completecoding region. However, for smaller quantities of mRNA a full lengthstrand may be only represented a few times.

[0003] As outlined in FIGS. 1a and 1 b, the current procedure alsorequires homopolymeric tailing of both the cDNA sequences and therestriction digested cloning vectors, thus doubling the amount ofmanipulation involved. In addition, homopolymeric tailing of the vectorresults in loss of the original restriction site thereby limiting theease of subsequent excision of the cloned cDNA region for the transferto other expression or amplification vectors.

[0004] Accordingly, improvements to simplify cloning of mRNA sequencesfor use in the cloning of cDNAs for expression of proteins and in theconstruction of cDNA libraries are desired.

SUMMARY OF THE INVENTION

[0005] In the present invention a simplified method of directionalcloning is provided. This method can be used, for example, in thecloning of the 5′ ends of cDNAs. The present invention differs fromprior art cloning methods requiring homopolymeric tailing of both cDNAsequences and restriction enzyme digested vectors along with completesecond strand synthesis before homopolymeric tailing. The method of thepresent invention improves the efficiency of the cloning of 5′-cDNA endsthereby increasing the likelihood of constructing full-length cDNAlibraries comprised of overlapping cDNA subsequences.

[0006] The present invention uses oligonucleotides encoding restrictionsites to create local double-stranded regions upon the first strand cDNAproduct of reverse transcriptase. The double-stranded regions arecleaved by double-strand requiring restriction endonucleases and serveto limit the regions to be replicated in a second (+) strand synthesis.Use of these oligonucleotides also increases the accuracy of replicationof the entire shorter (−) cDNA strand to yield more of the 5′ (+) cDNAsequences necessary for obtaining a full representation of the entiremRNA coding sequence.

[0007] The method of the present invention also uses an oligonucleotideprimer containing the same restriction site that is homopolymericallytailed to complement the homopolymerically tailed 3′ end of the (−) cDNAstrand. The 5′ end of this primer hybridizes to the palindromiccomplement 3′ end of the restriction digested (−) cDNA strand therebyforming a more stable and replication competent gapped single-strandedcircle. The resultant double-stranded product contains a unique copy ofthe targeting restriction site encoded by the priming oligonucleotide.Cleavage at this site yields double-stranded cDNA containing pairs thatcan be directly ligated into appropriate multiple cloning sites ofcommercial cloning and expression vectors. Since the restriction site ispreserved and flanks the cDNA insert, the desired cDNA sequences can bereadily excised and transferred to other vectors if necessary.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIGS. 1a and 1 b are schematics detailing the steps required byprior art procedures used to obtain cDNA clones from poly A+ mRNA. Acomplete set of clones containing different cDNAs representing allpossible coding sequences derived from isolated mRNAs constitute a cDNAlibrary. Regions of RNA, first strand (−) DNA and second strand (+) DNAare indicated by different stippling patterns in the bars. Thenucleotide sequences of homopolymeric tailings are indicated in boldtype. Steps are numbered sequentially as indicated. Those listed on theleft are for preparation of cDNA. Those listed on the right are forpreparation of the cloning vector.

[0009]FIGS. 2a, 2 b, and 2 c are schematics detailing the steps of themethod of the present invention when used for obtaining cDNA clones. Theleft portion of the figures show the method for cDNAs derived fromoligo-dT priming. The right portion of the figure shows the method forrandom priming of the poly A+ mRNA.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides a method of directional cloningwhich uses the 5′-ends of RNAs, for example, to obtain cDNA clones. Adetailed schematic of the method of the present invention being used toproduce cDNA clones is provided in FIGS. 2a-c. In this embodiment,oligo-dT or random priming of poly A+ mRNA is used to generate (−) firststrand cDNAs. These cDNAs are then homopolymerically tailed with dG ordC using terminal deoxynucleotidyl transferase. After tailing, theheteroduplex is denatured by heat and the mRNA removed by alkalinehydrolysis or RNAse digestion to yield single-stranded (−) cDNA. Thesingle-stranded (−) cDNA is the mixed with an first oligonucleotideincorporating a palindromic restriction site in the middle which isflanked on both the 5′ and 3′ sides with at least two completelydegenerate nucleotides. In a preferred embodiment the firstoligonucleotide consists of ten bases, including a 6 base palindromicrestriction site, such as that for EcoRI, flanked by two degenerativenucleotides, as the ten base overall length allows a high degree ofspecificity of targeting with reasonable annealing temperature. However,this oligonucleotide can be longer to incorporate other palindromicrestriction endonuclease recognition sequences. Examples of restrictionendonuclease recognition sequences which can be used include, but arenot limited to, BglII, ClaI, EcoRV, SacI, KpnI, SmaI, BamHI, XbaI, SalI,AccI, AvaI, PstI, SphI, HindIII, HincII, NsiI, NotI, SfiI, ApaI, NcoI,StuI, NdeI, PvuII, and XhoI. After mixing, the oligonucleotide-cDNAmixture is slowly cooled from 50° C. to 37° C. and the cognaterestriction enzyme is added. The resulting annealed, shortdouble-stranded DNA segments correspond to the positions of theserestriction sites on the (−) cDNA. Cleavage by the cognate restrictionenzyme yields single-stranded cDNAs bound on their 5′ end by the “stickyend” left by the restriction enzyme used and on their 3′ end by apoly-dG or −dC tract. Thus, the method of the present invention allowsspecific site-directed cleavage of the single-stranded (−) cDNA therebyeliminating the need for second strand synthesis of the entire (+) cDNAto provide the double-stranded restriction site as in prior art methods.Accordingly, the present invention is much simpler and requires lesstime than the prior art methods. Further, considerably smaller amountsof oligonucleotide triphosphate reagents are required.

[0011] A second oligonucleotide comprising nucleotides complementary tothe 3′ end of the cDNAs and containing the same restriction site as inthe first oligonucleotide is then annealed to the 3′ poly-dG or −dCtailed single-stranded (−) cDNA by a similar cycle of heating and slowcooling as described above. Since this single-stranded (−) cDNA containsthe cognate “sticky end” at its 5′ terminus, the 5′ end can loop backand also anneal to the second oligonucleotide at the restriction site.The resulting primed and gapped single-stranded (−) cDNA is stabilizedand rendered replication-competent for second strand synthesis of (+)cDNA by the addition of a DNA polymerase and DNA ligase. Since the cDNAregion to be replicated is shorter than the original full-lengthsequence, the likelihood of it being completely and accuratelyreplicated is increases over standard methods requiring the traverse ofa longer region of (−) cDNA. The resulting double-strandedclosed-circular cDNA is readily separated from linear single-strandedfragments and trinucleotides by spin column chromatography or agarosegel electrophoresis.

[0012] In addition, the resultant double-stranded cDNAs are readilylinearized with the cognate restriction enzymes to regenerate “stickyends”: compatible for direct ligation into vectors similarly linearized.This eliminates the extra effort in homopolymeric tailing of the vectorprior to insertion of the cDNA by prior art methods. Further, the methodof present invention preserves the cloning restriction site thusallowing ready excision of the desired cDNA sequences by the samerestriction enzyme.

[0013] As will be obvious to those of skill in the art upon reading thisdisclosure, the directional cloning method of the present can also beused in different embodiments. For example, by utilizing a complete setof oligonucleotides containing the common restriction sites utilized inthe multiple cloning sites of plasmid or phage vectors, the presentmethod can be used to provide a complete set of restriction sitedelimited cDNA sublibraries which would greatly facilitate both cloningand sequence analysis of cDNAs. A mRNA can be effectively scanned forall potential restriction sites thereby ensuring that a cDNA sublibrarywould encode the corresponding desired 5′ ends for almost all encodedmRNA.

1 4 1 10 DNA Artificial sequence Synthetic 1 nngaattcnn 10 2 10 DNAArtificial sequence Synthetic 2 nncttaagnn 10 3 11 DNA Artificialsequence Synthetic 3 gaattccccc c 11 4 10 DNA Artificial sequenceSynthetic 4 aattcccccc 10

What is claimed:
 1. An improved method of directional cloning of DNAcomprising annealing a first oligonucleotide encoding a restriction siteto single-stranded (−) cDNAs to create double-stranded regions on thesingle-stranded (−) cDNA so that regions to be replicated in a second(+) strand synthesis are limited.
 2. The method of claim 1 furthercomprising annealing a second oligonucleotide encoding the restrictionsite and having a homopolymeric tail complementary to a homopolymerictail at the 3′ end of the single-stranded (−) cDNAs to the 3′ end of thesingle-stranded (−) cDNA strand to form a stable replication competentgapped single-stranded circle by hybridization of the restriction siteof the second oligonucleotide to the restriction site of thesingle-stranded (−) cDNAs.